P40. Analysis of acetylation in oocytes and 1-cell embryos

Abstract

Major mechanisms of epigenetics are post-translational histone modifications such as reversible acetylation. Histone deacetylases (HDACs) play critical roles in regulation of histone acetylation but also act on non-histone substrates. It is reported that the developmental rate of cloned embryos is significantly improved by treatment with trichostatin A (TSA), a HDAC inhibitor. In contrast, the developmental rate of fertilized embryo is decreased after TSA treatment. Currently it has been assumed that TSA treatment induces hyperacetylation of histones which changes gene regulations. However, TSA treatment can also have impact on acetylation level of non-histone proteins, for example p53 and tubulin which may contribute to the embryonic development. To elucidate the functions of HDAC in the early development and reprogramming it is important to clarify the acetylation states of these non histone proteins in TSA-treated embryos. In this study, we investigated the acetylation levels in unfertilized oocytes as well as in vitro fertilized (IVF) embryos and parthenogenetic developed (PG) embryos after 10 h activation by immunostaining using anti-acetylated-Lysine antibody and anti-acetylated-alpha-Tubulin antibody. Our results showed hyperacetylation in pronuclei of IVF embryos and PG embryos. Additionally, by TSA treatment, acetylation of pronuclei was accelerated by over 1.5 times and that of cytoplasm was accelerated by over 2 times in both IVF embryos and PG embryos. Interestingly, comparison of the acetylation levels between male and female pronuclei in IVF embryos revealed acetylation level of male pronuclei significantly higher than that of female pronuclei, and this difference remained even with TSA treatment. Furthermore, we have shown that acetylation of tubulin is accelerated by both activation and TSA treatment. These results show that the acetylation state of embryos, including histone and non-histone proteins, was changed by activation. Especially, we have demonstrated that alpha-tubulin which constructs spindle in MII oocytes also constructs cytoskeleton in IVF embryos and PG embryos by activation. In addition, our results suggest that there is the possibility that the acetylation level of non-histone proteins also accelerated by the acetylation level of cytoplasm. Furthermore, the difference of the acetylation level between male and female pronuclei was not affected by HDAC activity because the difference was not cancelled out by TSA treatment.